Neurons of the central nervous system transmit information via electrical impulses. These impulses are generated by electrochemical potentials caused by the movement of charged particles across (i.e. through) the cell membrane. The size of the impulse transmitted is a function of the membrane conductance The term "conductance," as applied to transmission of ionic charge across a cell membrane, means the incremental current (or current-like) response exhibited through the cell membrane as a result of the application of a voltage (an incremental change in the electric field) across the membrane.
A calcium channel is a structure that spans the thickness of the lipid bilayer in cell membranes and allows calcium ions to move passively across this lipid bilayer according to the calcium electrochemical gradient between the interior of the cell (where calcium concentration is lower) and the extra-cellular fluid (where calcium concentration is higher). These channels demonstrate a higher specificity for the movement of the calcium ion across this channel-like structure, although a calcium channel's selectivity with respect to other divalent cations is never perfect. Calcium channels can be activated by changes in the electric field of the cell membrane.
A calcium pump is a different structural moiety (probably one or more macromolecules) of the cell membrane. This moiety spans the cell membrane and produces an active movement of calcium ions against their electrochemical gradient. Ion pumps are therefore different from ion channels in that they require energy to generate ionic movement since ionic flow through a pump occurs against the electrochemical gradient.
In a resting state, the interior of a nerve cell is negatively charged with respect to the extracellular medium or environment. This difference in potential, which is observed across the cell membrane, is reversed when an impulse passes along the nerve. For a brief period of time, the polarity of the nerve cell becomes positive. This sequence of events is known as an "action potential."
Direct stimulation of neurons in vitro produces action potentials having two main components: a fast spike, due to sodium conductance, and a slower, calcium-dependent spike (Llinas, R. and Sugimori, M. J. Physiol. 305:197-213 1980). Sodium-dependent potentials are the prominant feature of the somatic (or cell-body) response, while calcium-dependent action potentials are more apparent in the dendrites.
Llinas and Yarom (J. Physiol. 315: 549-567; J. Physiol. 315: 569-584, 1981) have previously disclosed that guinea-pig inferior olivary (I.O.) cells from the brainstem exhibit a calcium-dependent conductance which also has two components. The sodium- and calcium-dependent conductance is illustrated in FIG. 1. Using techniques described below, the authors found that stimulation of these cells in vitro generates action potentials comprising a fast spike, 1 (due to sodium conductance) followed by an after-depolarization potential (ADP), 2. The ADP was shown to be due to the activation of a high-threshold calcium conductance (HTCC). The ADP was followed by an after-hyperpolarization potential (AHP), 3. In turn, the AHP was followed by a rebound depolarization spike, 4. The rebound spike (RS) was shown to be due to the activation of low-threshold calcium conductance (LTCC). The shaded areas in FIG. 1 represent the two types of voltage spikes due to the two types of calcium conductance. The ADP and AHP are tetrodo-toxin-insensitive (textrodoxin inhibits sodium conductance) and, therefore, they cannot be due to sodium conductance. The following can be considered an operative definition of the RS (or LTC spike):
The RS is generated by the presence of LTCC as follows:
(a) Following tetrodoxin poisoning of the cell membrane, the RS occurs as the membrane is abruptly depolarized with square voltage pulses of increasing amplitude, if the membrane potential is more negative than -70 mV. The threshold in the inferior olivary cells studied by the present invention is 65 mV.
(b) The RS occurs as a rebound action potential following hyperpolarization of the cell membrane from a resting potential. The firing level of the RS spike for the inferior olivary cells studied by the present inventors is the same as in (a), i.e. -65 mV.
(c) When a voltage clamp technique (described below) is used, the RS generates an ionic current that occurs at negative values of cell membrane potential, rises to a maximum, and is then inactivated--also within the negative potential range (at about -45 mV for the I.O. cells studied).
HTCC is involved in dendridic action potentials, in certain components of heart action, and in synaptic transmission. LTCC appears to be a somatic response.
Tremor consists of a more or less regular rhythmic oscillation of a part of the body about a fixed point. The rate of this oscillation varies from individual to individual but, in a particular patient, the rate is fairly constant in all affected parts of the body. Tremor may be caused by specific pathological diseases (e.g. Parkinson's Disease) or be due to specific lesions in the central nervous system, or it may be of unknown origin.
From a functional point of view, tremor can be seen as a modification of the basic electrophysiological properties of cells comprising the central nervous system.
It has been suggested (Llinas, R.R. in Movement Disorders: Tremor, pp. 165-182, Findley, L. J. and Capildeo, R., eds., MacMillan, 1984; incorporated by reference) that the interplay between the high-threshold (or dendritic) calcium conductance and the low-threshold (or somatic) conductance could result in central oscillatory properties of nerve cells which have the same cyclic rhythmic frequence such as that found in physiological and abnormal tremor as well as in Parkinson's tremor.
The present inventors have surprisingly found that certain compounds (such as aliphatic alcohols) at extremely small amounts are capable of blocking (partially or completely) the low threshold calcium conductance (which generates the so-called rebound calcium spike). Significantly, these compounds, when used in small amounts, do not affect the high-threshold calcium conductance. Furthermore, the inventors have found that although lower alkyl alcohols have a blocking effect, the higher alcohols do so at extraordinarily low concentrations.
The prior art contains several references purporting to describe the effects of alcohols on calcium conductance, and in particular the effects of ethanol. In all instances known to the present inventors, however, the alcohols were used for a different purpose (to block HTCC, i.e., as "anaesthetics") and in amounts markedly exceeding those of the present invention.
Requena et al. (J. Gen. Physiol. 85: 789-804. 1985) disclose that exposure of squid axons to octanol, at a concentration of 10.sup.-4 M, correlated with an apparent increase in the observed intracellular calcium concentration in these axons. In other words, Requena et al state that octanol interferes with the ability of the calcium ions to leave the cell by crossing the cell membrane. This phenomenon is unrelated to blockage or non-blockage of calcium channels (high- or low-threshold).
As explained above, a calcium channel is a passive transport mechanism by which calcium ions move down their electrochemical gradient. In all cells, calcium concentration is low inside the cell (e.g., 10.sup.-7 M) and high in the extracellular medium (e.g., 10.sup.-3 M) and so a calcium channel allows calcium to go into the cell.
By contrast, outward calcium transport takes place via "a calcium pump," an entirely different mechanism which transports calcium against a concentrational gradient (from the low concentration inside to the high concentration outside). An ion pump is therefore an active membrane structure, usually an enzyme (e.g., sodium ATPase) which requires energy (ATP: adenosine triphosphate) to carry ions across the membrane. Requena et al anaesthetize the cells and, therefore, "paralyze" the pump mechanism. In any event, Requena's observations concern a totally different phenomenon from that of the present invention and require different (markedly higher) octanol concentrations. If the present inventors measured the intracellular calcium concentration after exposure of neuron cells to the alcohol in accordance with the present invention, they would observe a normal, or a lower-than-normal intracellular calcium concentration, (i.e. an effect opposite to that said to have been observed by Requena et al.), which would be due to inability of calcium to enter the cell through the blocked Ca channel. Furthermore, investigations conducted by the present inventors revealed no evidence of the presence of a low-threshold calcium channel in squid axons (unpublished observation).
Similarly, Leslie et al. (J. Pharm. Exp. Ther. 225:571-575. 1983) disclosed that ethanol, at 2.5.times.10.sup.-2 -1.5.times.10.sup.-1 M, inhibited voltage-dependent calcium uptake into synaptosomes isolated from rat brains. Aside from the high ethanol concentrations said to be used, there was no mention in this publication of studies on low-conductance calcium channels.
Michaels et al. (Biochem. Pharm. 32:963-969, 1983) described the effects of ethanol (10.sup.-1 M), propanol (10.sup.-2 M) and butanol (10.sup.-1 M) on calcium-dependent fluxes in rat brain synaptic membrane vesicles. All three alcohols inhibited calcium influxes in this experimental system.
Kitagawa et al. (Biochem. Biophys. Acta 798:210-215, 1984) disclose the use of butanol (5.times.10.sup.-2 M) or hexanol (5.times.10.sup.-3) as an inhibitors of calcium mobilization in bovine platelets. The mechanism in this case is a calcium pump similar to that studied by Requena et al and the authors of the other papers described above. Moreover, the LTCC has not been demonstrated in platelets, and the mechanism by which calcium enters platelets is not known.
It has been noted in the past that one or two drinks of an alcoholic beverage can abate the symptoms of familial tremor temporarily: Harrison's Principles of Internal Medicine, p. 96 Isselbacher, K. J. et al. eds. McGraw-Hill, New York, N.Y. 1980. One or two drinks of an alcohol-containing beverage would produce blood levels approximately on the order of 10.sup.-2 M in ethanol, a concentration notably higher than that necessary in the present invention. Furthermore, the intoxicating and addictive properties of ethanol are well-known.
In addition, the aforementioned empirical observations were never correlated with LTCC nor with the central nervous system. For these reasons, the above-cited phenomenon has only superficial, if any, relevance to the present invention.
Current treatment for tremors comprises administration of beta-adrenergic blockers (such as propranolol hydrochloride, and its derivatives). These drugs act via a mechanism totally different from that of the present invention, and affect muscle cells as opposed to neuron cells. Beta-adrenergic blockers cause a myriad of side-effects (e.g. bronchodilation, lightheadedness, bradycardia, hallucinations, and kidney and liver abnormalties). In addition, these drugs are not effective in all patients and are harmful to some (e.g. asthmatics). Use of the present invention should lead to minimal side-effects due to the very low concentration of alcohols administered. At the preferred alcohol concentrations, primarily only LTCC would be affected and a larger patient population could be treated using the present invention instead of beta-adrenergic blockers. Furthermore, use of alcohols in accordance with the present invention could be made in conjunction with use of beta-blockers.